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Natural Single-Nucleotide Deletion in Chymotrypsinogen C Gene Increases Severity of Secretagogue-Induced Pancreatitis in C57BL/6 Mice

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Natural Single-Nucleotide Deletion in Chymotrypsinogen C Gene Increases Severity of Secretagogue-Induced Pancreatitis in C57BL/6 Mice Natural single-nucleotide deletion in chymotrypsinogen C gene increases severity of secretagogue-induced pancreatitis in C57BL/6 mice Andrea Geisz, … , Eszter Hegyi, Miklós Sahin-Tóth JCI Insight. 2019;4(14):e129717. https://doi.org/10.1172/jci.insight.129717. Research Article Gastroenterology Inflammation Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast with trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC because of a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the Ctrc+ strain, the severity of cerulein- induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation, suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. Taken together with prior human genetic and biochemical studies, the observations provide conclusive evidence for the protective role of CTRC against pancreatitis. Find the latest version: https://jci.me/129717/pdf RESEARCH ARTICLE Natural single-nucleotide deletion in chymotrypsinogen C gene increases severity of secretagogue-induced pancreatitis in C57BL/6 mice Andrea Geisz,1 Zsanett Jancsó,1 Balázs Csaba Németh,1 Eszter Hegyi,1 and Miklós Sahin-Tóth1,2 1Center for Exocrine Disorders, Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston University, Boston, Massachusetts, USA. 2Department of Surgery, UCLA, Los Angeles, California, USA. Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast with trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC because of a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the Ctrc+ strain, the severity of cerulein- induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation, suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. Taken together with prior human genetic and biochemical studies, the observations provide conclusive evidence for the protective role of CTRC against pancreatitis. Introduction The inflammatory disease continuum of the pancreas starts with a sentinel attack of acute pancreatitis, fol- lowed by recurrent acute pancreatitis attacks with eventual progression to chronic pancreatitis (1). The patho- logical drivers underlying the inflammatory process are frequently genetic mutations that promote the prema- ture, intrapancreatic activation of the digestive protease trypsin (2). The high-impact susceptibility genes that influence activation of trypsinogen to trypsin includePRSS1 (serine protease 1, cationic trypsinogen), SPINK1 (serine protease inhibitor Kazal type 1) and CTRC (chymotrypsin C). Gain-of-function mutations in PRSS1 promote trypsinogen activation whereas loss-of-function mutations in SPINK1 and CTRC impair protective trypsin inhibition and trypsinogen degradation, respectively (2). CTRC is a minor chymotrypsin isoform capable Authorship note: AG and ZJ of degrading trypsinogen and thereby suppressing its activation to trypsin (2–4). The clinically most common contributed equally to this work. PRSS1 mutations cause hereditary pancreatitis by rendering trypsinogen resistant to protective degradation by Conflict of interest: The authors have CTRC and thereby increasing intrapancreatic trypsin activity (2, 4). Although the principal action of CTRC is declared that no conflict of interest to promote trypsinogen degradation, it also enhances trypsinogen activation by processing the cationic tryp- exists. sinogen activation peptide to a shorter form, which is more sensitive to trypsin-mediated activation (4–6). This Copyright: © 2019 American Society minor effect becomes important only when certain cationic trypsinogen mutations (e.g., p.A16V) hijack this for Clinical Investigation mechanism and thereby stimulate trypsinogen activation to a pathological extent (4, 5, 7). A defensive role Submitted: April 22, 2019 for chymotrypsin-mediated trypsinogen degradation was further corroborated by the recent observation that a Accepted: June 13, 2019 commonly occurring 16.6-kb inversion at the human CTRB1-CTRB2 locus (encoding chymotrypsin B1 and B2) Published: July 25, 2019. has a small but significant effect on pancreatitis risk (8). The minor inversion allele alters the expression ratio Reference information: JCI Insight. of the 2 isoforms in such a manner that trypsinogen degradation becomes more efficient, resulting in a small 2019;4(14):e129717. https://doi. protective effect against pancreatitis. Taken together, human genetic and biochemical evidence indicates that org/10.1172/jci.insight.129717. chymotrypsin-dependent trypsinogen control is essential for pancreas health and protection against pancreatitis. insight.jci.org https://doi.org/10.1172/jci.insight.129717 1 RESEARCH ARTICLE Figure 1. Expression of CTRC in the mouse pancreas. (A) Expression of CTRC protein in the pancreas of 7 inbred mouse strains and the Ctrc+ strain. Pancreas homogenates (10 μg total protein) from the indicated mice were analyzed by Western blotting. ERK1/2 pro- tein was measured as loading control. (B) Expression of Ctrc mRNA in the pancreas of 7 inbred mouse strains and the Ctrc+ strain, determined by RT-PCR and agarose gel electrophoresis. 18S rRNA was measured as loading control. Representative gels from 2 inde- pendent experiments are shown. To confirm the notion that loss of chymotrypsin function increases intrapancreatic trypsin activation and worsens pancreatitis, we recently generated a mouse strain deficient in CTRB1, the major chymo- trypsin isoform in mice (9). Previously, using purified proteins, we demonstrated that mouse trypsino- gens may be regulated by mouse CTRC and CTRB1 through degradation and regulatory cleavages that suppress activation (10). When given repeated injections of supramaximal stimulatory doses of cerulein, Ctrb1-del mice developed severer pancreatitis than C57BL/6N control mice. Importantly, intrapancreatic trypsin activation was significantly elevated inCtrb1-del mice, confirming the protective role of chymo- trypsin-dependent trypsinogen degradation (10). Although these findings were important conceptual- ly, they did not address the specific role of CTRC, the chymotrypsin isoform more relevant to human disease. Therefore, in the present study we set out to characterize CTRC expression in different mouse strains with the intent of correlating experimental pancreatitis responses to pancreatic CTRC levels. Sur- prisingly, we found that the popular inbred C57BL/6 mice did not express functional CTRC because of a natural single-nucleotide deletion. We restored a functional Ctrc gene in C57BL/6N mice and studied experimental pancreatitis responses in the Ctrc+ strain. The results presented below offer long-awaited animal model evidence for the predicted protective role of CTRC in pancreatitis. Results C57BL/6 mice are naturally deficient in CTRC. To investigate whether variations in CTRC expression affect pancreatitis severity, first we compared CTRC protein levels in the pancreas of 7 inbred mouse strains. Unexpectedly, we found that C57BL/6 mice, including both the N and J substrains, expressed no detect- able CTRC protein, as judged by Western blotting of pancreas extracts. In contrast, all other mice tested contained comparable levels of pancreatic CTRC protein (Figure 1A). Consistent with the protein expres- sion defect, reverse transcription PCR (RT-PCR) analysis of Ctrc mRNA in the pancreas of C57BL/6 mice yielded 2 abnormal products. DNA sequencing revealed that the faint larger band contained a single nucleotide deletion (c.117delT) and the more prominent smaller band a 98-nucleotide deletion correspond- ing to exon 3 (Figure 1B). As expected, RT-PCR amplicons from the pancreas of the other mouse strains showed the expected size and a DNA sequence with no deletion (Figure 1B). We confirmed the c.117delT deletion in exon 2 of the Ctrc gene on chromosome 4 in the National
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